Exploratory biomarker analysis in the phase III L-MOCA study of olaparib maintenance therapy in patients with platinum-sensitive relapsed ovarian cancer

Background The prospective phase III multi-centre L-MOCA trial (NCT03534453) has demonstrated the encouraging efficacy and manageable safety profile of olaparib maintenance therapy in the Asian (mainly Chinese) patients with platinum-sensitive relapsed ovarian cancer (PSROC). In this study, we report the preplanned exploratory biomarker analysis of the L-MOCA trial, which investigated the effects of homologous recombination deficiency (HRD) and programmed cell death ligand 1 (PD-L1) expression on olaparib efficacy. Methods HRD status was determined using the ACTHRD assay, an enrichment-based targeted next-generation sequencing assay. PD-L1 expression was assessed by SP263 immunohistochemistry assay. PD-L1 expression positivity was defined by the PD-L1 expression on ≥ 1% of immune cells. Kaplan–Meier method was utilised to analyse progression-free survival (PFS). Results This exploratory biomarker analysis included 225 patients and tested HRD status [N = 190; positive, N = 125 (65.8%)], PD-L1 expression [N = 196; positive, N = 56 (28.6%)], and BRCA1/2 mutation status (N = 219). The HRD-positive patients displayed greater median PFS than the HRD-negative patients [17.9 months (95% CI: 14.5–22.1) versus 9.2 months (95% CI: 7.5–13.8)]. PD-L1 was predominantly expressed on immune cells. Positive PD-L1 expression on immune cells was associated with shortened median PFS in the patients with germline BRCA1/2 mutations [14.5 months (95% CI: 7.4–18.2) versus 22.2 months (95% CI: 18.3–NA)]. Conversely, positive PD-L1 expression on immune cells was associated with prolonged median PFS in the patients with wild-type BRCA1/2 [20.9 months (95% CI: 13.9–NA) versus 8.3 months (95% CI: 6.7–13.8)]. Conclusions HRD remained an effective biomarker for enhanced olaparib efficacy in the Asian patients with PSROC. Positive PD-L1 expression was associated with decreased olaparib efficacy in the patients with germline BRCA1/2 mutations but associated with improved olaparib efficacy in the patients with wild-type BRCA1/2. Trial registration NCT03534453. Registered at May 23, 2018. Supplementary Information The online version contains supplementary material available at 10.1186/s12916-024-03409-9.


Background
Ovarian cancer remains one of the most difficult-totreat malignancies, with 313,959 new cases and 207,252 cancer-related deaths annually worldwide [1].Due to the insidious onset of ovarian cancer, over 70% of patients with the disease are diagnosed with an advanced stage, thus having abysmal prognosis [1].Though the initial response rate to platinum-based chemotherapy exceeds 70%, most patients will inevitably experience recurrence [2].In the treatment of platinum-sensitive relapsed ovarian cancer (PSROC), the application of poly (ADP-ribose) polymerase (PARP) inhibitors is one of the most encouraging advances [3].
PARP inhibitors enable synthetic lethality in tumour cells with homologous recombination deficiency (HRD), especially those with mutated BRCA1/2 (BRCAm) [4].As the first authority-approved PARP inhibitor, olaparib prolongs progression-free survival (PFS) among patients with PSROC in Study 19 regardless of BRCA1/2 mutation status and extends PFS among BRCAm patients in SOLO2 trial [5,6].Furthermore, OPINION trial has demonstrated that HRD-positive patients with PSROC exhibited improved responses to olaparib compared to their HRD-negative counterparts [7].Owing to the paucity of clinical trials investigating olaparib efficacy in the Asian patients, we initiated the prospective phase III multi-centre L-MOCA study (NCT03534453) to assess the efficacy of olaparib exclusively in the Asian (mainly Chinese) patients with PSROC and its association with mutations of BRCA1/2 and homologous recombination repair (HRR) genes [8].The association between HRD and treatment benefits of olaparib maintenance therapy in the Asian patients with PSROC remains unclear.
In addition to eliciting targeted tumour-killing effects, PARP inhibitors could modulate tumour immune microenvironment.The efficacy of PARP inhibitors partially depends on anti-tumour immune responses [9].DNA damage caused by PARP inhibition increases tumour mutational load and neoantigen expressions, thereby inducing anti-tumour immune responses through recruiting cytotoxic T lymphocytes (CTLs) [9].Meanwhile, PARP inhibitors could upregulate programmed cell death ligand 1 (PD-L1) expression on tumour cells, making the combination of PARP inhibitors and immune checkpoint inhibitors a scientifically rational approach [10].Moreover, BRCAm tumours display enhanced infiltration of CTLs than HRD-negative tumours [11].Therefore, it is intriguing to decipher the effects of PD-L1 expression, an immunologic marker, on PARP inhibitors efficacy under different genotypes.
Accurate detection of genetic vulnerabilities of cancers could foster the personalised use of targeted therapies.Compared with traditional biopsy, liquid biopsy is advantageous in minimally invasive sampling, fewer contraindications and complications, and comprehensive characterization of the mutation landscape [12].Circulating tumour DNA (ctDNA) carries the molecular aberrations of the primary tumours and effectively detects tumour-specific mutations that could be exploited by targeted therapies [13].However, the consistency between ctDNA and tumour samples to detect molecular abnormalities in ovarian cancer, especially the evidence derived from prospective clinical trials, remain underexplored.
The prospective phase III multi-centre L-MOCA study was the first trial to evaluate olaparib efficacy and tolerability exclusively in the Asian patients with PSROC.The PFS and subgroup analysis by BRCA1/2 and HRR mutation status were previously released [8].In this study, the preplanned exploratory biomarker analysis of the L-MOCA trial was conducted to evaluate other potential biomarkers of treatment benefits with olaparib including HRD and PD-L1 expression and to investigate the ability of ctDNA to detect BRCA /HRR mutations.

Study design and patients
This study was the preplanned exploratory biomarker analysis of the international, prospective, open-label, single-arm, phase III L-MOCA trial, which recruited 225 patients from 28 centres in China (N = 22) and Malaysia (N = 6) between March 2018 and December 2020.The inclusion and exclusion criteria have been described in the previous report [8].Patients aged 18 years or older, with the Eastern Cooperative Oncology Group performance status score of 0 or 1, who had received at least two courses of platinum-based chemotherapy, and had achieved either a complete or partial radiological response defined by the Response Evaluation Criteria in Solid Tumours (RECIST) guidelines were included.Patients were excluded if they had been exposed to PARP inhibitors.Eligible patients received oral olaparib (300 mg) twice daily until disease progression or unacceptable toxicity.As a committed study for registration, the L-MOCA trial has strict data quality control with the rate of source data verification reaching 100%.Approved by the national regulatory authority and the respective local ethics committees at each participating institution [( 2018)-(3)-No.4for the leading site], this study was conducted following the Good Clinical Practice guidelines and the Declaration of Helsinki.Informed consent was obtained from all individual participants included in the study.The trial protocol is presented in Additional file 1.

Study endpoints and assessments
The primary endpoint of the L-MOCA study was investigator-assessed PFS according to RECIST 1.1 criteria, defined as the time from the date of first olaparib dose to the date of disease progression or death.Clinical and objective radiologic tumour assessments were conducted at baseline, every 12 weeks until week 72, and thereafter every 24 weeks until objective disease progression.Results of the primary analysis of PFS, along with safety, have been published [8].Secondary endpoints included overall survival, time from first dose to second progression, and others detailed in the trial protocol (Additional file 1).This study reported the exploratory objective of exploring biomarkers in tumour tissues or blood predictive of sensitivity to olaparib including PD-L1 expression, HRD status, and BRCA /HRR mutation status in the ctDNA.

Sample collection and DNA extraction
For patients with biomarker informed consent, their whole blood and tumour samples were collected at any time following diagnosis but prior to study entry.Tissues with tumour proportions ≥ 10% and necrotic proportions ≤ 50% were selected for DNA extraction.Tumour DNA and ctDNA were extracted from tumour tissue and plasma, respectively, using the QIAamp DNA Formalin-fixed paraffin-embedded (FFPE) Tissue Kit (Qiagen, Valencia, CA) and QIAamp Circulating Nucleic Acid Kit (Qiagen).Germline DNA was obtained from paired white blood cells (WBCs) via the QIAamp DNA Blood Mini Kit (Qiagen).DNA quantification was performed by a Qubit 2.0 fluorimeter (Thermo Fisher Scientific).

BRCA /HRR/HRD status analysis
For each patient, we conducted paired genetic testing of tumour samples, ctDNA, and germline DNA derived from peripheral WBCs.Germline and somatic BRCA mutation status were established using DNA extracted from WBCs and FFPE tumour samples, respectively.To validate the BRCA status, 5% was set to limit the detection of BRCA single-nucleotide variant (SNV) or insertion/deletion (INDEL).Next-generation sequencing (NGS) on a panel of 72 genes were performed to determine the HRR alterations for both tumour and ctDNA samples.The details of HRR genes were presented in the previous report [8].Sequence data were mapped to the reference human genome (UCSC hg19) using Burrows-Wheeler Aligner (BWA-MEM, v.0.7.10).Local alignment optimization and variant calling were performed using Genome Analysis Tool Kit (GATK, v.3.3) and Var-Scan v.2.4.3, respectively.Variants were filtered using the VarScan filter pipeline, where loci with depth < 100 were filtered out to eliminate benign variants.Base-calling in samples required at least 8 supporting reads for SNV and 5 supporting reads for INDEL variations.Variants with population frequency over 0.1% in the ExAC, 1000 Genomes, dbSNP, or ESP6500SI-V2 databases were grouped as single-nucleotide polymorphisms (SNPs) and excluded from further analysis.The remaining variants were annotated with ANNOVAR (2016-02-01 release) and SnpEff v.3.6.Xcavator software was applied to analyse DNA translocation.Copy number variations (CNVs) were analysed based on the depth of coverage data of capture intervals using an in-house developed algorithm.The limit of detection for CNVs was 1.5 for deletions and 2.64 for amplification, respectively.Classifications of all detected gene variants were based on the American College of Medical Genetics recommendations for standards of interpretation and reporting of sequence variants.Variants were classified as pathologic, likely pathologic, variation of uncertain significance, likely benign, or benign [14].Deleterious BRCA1/2 and HRR mutations included pathologic and likely pathologic mutations and were defined as detailed functional defects or changes in gene expression.
When analysing the ctDNA results, mutations from both germline and somatic origins were identified.The mutations were categorised based on their presence or absence in the WBCs.If a mutation was present in both the ctDNA and WBCs, it was considered germline; if a mutation was solely present in the ctDNA, it was deemed somatic.

PD-L1 expression analysis with immunohistochemistry (IHC)
The Ventana SP263 assay (Ventana Medical Systems Inc.) was adopted to assess PD-L1 expression in FFPE tumour samples.Briefly, 5 μm sections were cut and stained for SP263 on the Ventana Benchmark Ultra platform according to the standard protocol.Subsequently, stained sections were scanned using Ventana iScan HT and scored based on the percentage of tumour cells and immune cells showing membranous positivity.PD-L1 evaluation was performed by pathologists who routinely use clone SP263 in their clinical practice.For each case, the highest scoring value across the scores was used.PD-L1 expression on immune cells (IC) and tumour cells (TC), and the combined positive score (CPS) were evaluated and calculated in accordance with previous literature [16].SP263 assay has been demonstrated to have similar levels of analytic performance with other FDA-approved PD-L1 IHC assays including 22C3 and 28-8 assays [16,17], with advantages of high sensitivity and relatively low cost in China, which contributed to most of the study subjects and where the PD-L1 analysis was performed centrally.

Statistical analysis
Data were summarised using descriptive statistics since the first dose of olaparib.Continuous variables were represented in mean ± standard deviation or median and interquartile range.Categorical variables were represented in frequency and percentages.PFS was estimated using the Kaplan-Meier method.All the analyses were performed using the SAS software, version 9.4.

Prevalence and patterns of PD-L1 expression
PD-L1 expression by IC, TC, and CPS was evaluated among 196 (89.5%) patients (Additional file 2: Table S4).PD-L1 was stained positive on ≥ 1% of IC in 28.6% of patients (N = 56), whilst PD-L1 was stained positive on < 1% of TC in the majority of patients (N = 180; 91.8%).For CPS, a scoring algorithm that considered both IC and TC, 25.5% of patients (N = 50) exhibited a CPS of ≥ 1. IHC staining showed that PD-L1 was dominantly expressed on IC (Additional file 2: Fig. S1A) and PD-L1 expression on IC contributed the most to CPS (Additional file 2: Fig. S1B, C).Therefore, PD-L1 expression on ≥ 1% of IC was set as the threshold for PD-L1 expression positivity in further analysis.
Interestingly, the effects of PD-L1 expression on olaparib efficacy disappeared in the HRD-positive subgroup.The mPFS did not differ between patients with negative and positive PD-L1 expression [16.6 months (95% CI: 8.6-22.2) versus 16.5 months (95% CI: 10.8-NA), Fig. 3C] in the HRD-positive subgroup.In the HRD-negative subgroup, positive PD-L1 expression was still associated with improved mPFS [16.5 months (95% CI: 2.6-NA) versus 8.3 months (95% CI: 6.9-13.3);Fig. 3C], but the number of PD-L1-positive patients in the HRD-negative subgroup was limited (N = 5).To elucidate the reasons underlying the association between PD-L1 expression and olaparib efficacy in gBRCA mutation subgroups and why the trend disappeared in the HRD-positive subgroup, we reviewed the demographics (Additional file 2: Tables S5, S6), pathology and extent of disease (Additional file 2: Tables S7, S8), and previously received anti-tumour therapies and responses (Additional file 2: Tables S9, S10) of PD-L1-positive and PD-L1-negative patients in gBRCA mutation and HRD subgroups.However, no contributing factor was found.The similar PFS between Fig. 1 Study workflow.We included 225 patients with PSROC from the phase III L-MOCA trial, among which 219 patients with signed biomarker informed consent were included for the biomarker analysis.After strict quality control processes, PD-L1 expression and HRD status were evaluated in 196 and 190 participants, respectively.BRCA /HRR mutations were assessed in all the tumour samples and 217 blood samples (circulating tumour DNA).Abbreviations: PSROC, platinum-sensitive relapsed ovarian cancer; PD-L1, programmed cell death ligand 1. HRD, homologous recombination deficiency; HRR, homologous recombination repair Fig. 2 Kaplan-Meier estimates of progression-free survival in HRD/BRCA subgroups.A Kaplan-Meier plots of progression-free survival analysis in HRD-positive, HRD-negative, and HRD-unknown patients.B Kaplan-Meier plots of progression-free survival analysis in HRD-positive BRCAm, HRD-positive BRCAwt, and HRD-negative patients.Abbreviations: HRD, homologous recombination deficiency; 95% CI, 95% confidence interval; BRCAm, BRCA1/2 mutated; BRCAwt, BRCA1/2 wild type PD-L1-positive and PD-L1-negative patients in the HRD-positive subgroup could be attributed to the similar proportion of gBRCAm (N = 56; 44.8%) and BRCAwt patients (N = 60; 48.0%) in this subgroup (Additional file 2: Table S11).

Discussion
In this preplanned exploratory biomarker analysis of the prospective phase III L-MOCA trial, we demonstrated that the HRD-positive patients with PSROC derived greater PFS benefits from olaparib maintenance therapy (See figure on next page.)Fig. 3 The association between PD-L1 expression and olaparib efficacy.A Kaplan-Meier plots of PFS analysis in PD-L1-negative (IC < 1%) and PD-L1-positive (IC ≥ 1%) patients.B Kaplan-Meier plots of PFS analysis in gBRCAm PD-L1-negative, gBRCAm PD-L1-positive, BRCAwt PD-L1-negative, and BRCAwt PD-L1-positive patients.C Kaplan-Meier plots of PFS analysis in HRD-positive PD-L1-negative, HRD-positive PD-L1-positive, HRD-negative PD-L1-negative, and HRD-negative PD-L1-positive patients.Abbreviations: PFS, progression-free survival; PD-L1, programmed cell death ligand 1; 95% CI, 95% confidence interval; gBRCAm, germline BRCA1/2 mutations; BRCAwt, wild-type BRCA1/2; HRD, homologous recombination deficiency Fig. 3 (See legend on previous page.) compared to the HRD-negative patients in an Asian population.Meanwhile, remarkable clinical outcomes were observed in HRD-positive patients harbouring BRCA1/2 mutations.Positive PD-L1 expression on immune cells was associated with decreased olaparib efficacy in gBR-CAm patients but associated with improved olaparib efficacy in BRCAwt patients.The prevalence of HRD in this cohort was 65.8%, being comparable to the percentage (68.7%)reported in the real-world study among the Chinese population [18,19] but higher than the widely accepted approximately 50% in patients with high-grade serous ovarian cancer [20].Besides, the incidence of gBRCA1/2 mutations was high in this cohort.The high prevalence of HRD and gBRCA1/2 mutations may be attributable to the enrichment effects of the platinum-sensitive recurrence setting on gBRCA1/2 mutations and HRD and the increased prevalence of gBRCA1/2 mutations in the  Chinese patients with ovarian cancer [18,[21][22][23].HRD has been recommended as a predictive biomarker for PARP inhibitors benefits in prospective setting as maintenance therapy in recurrent ovarian cancer, especially in the first line [22,[24][25][26][27].However, the evidence derived from the Asian patients has been scarce.Our study for the first time demonstrates that HRD is indeed associated with enhanced olaparib efficacy in the Asian patients with PSROC, thereby complementing and reinforcing the global evidence that links HRD with enhanced olaparib efficacy.
Currently, there is no standard threshold to define PD-L1 positivity in ovarian cancer, whether in terms of the percentage of cells stained positive or the type of cells considered (IC, TC, or both) [28].We unravelled that in the Asian patients with PSROC, PD-L1 was prevalently expressed on IC, which aligned with previous literature stating that most PD-L1 expression was observed in inflammatory infiltrate [29] or stromal tumour-infiltrating lymphocytes [30].However, the mechanisms underlying this pattern of PD-L1 expression in ovarian cancer remain underexplored.Using IC ≥ 1% as the threshold for PD-L1 expression positivity, we reported the previously uncharacterised association between PD-L1 expression and olaparib efficacy.Positive PD-L1 expression was associated with impaired olaparib efficacy in gBRCAm/ HRRm patients but associated with improved olaparib efficacy in BRCAwt/HRRwt population.Those data indicate that genetic profiling of gBRCA mutations, when aided by an easily accessible PD-L1 IHC assay, might enable a more accurate estimation of olaparib efficacy in clinical practice, paving the way for clinical trials exploring the efficacy of PARP inhibitors plus PD-1/PD-L1 blockade with gBRCA1/2 mutations and PD-L1 expression as stratification factors.However, these results warranted further investigations in future clinical trials with larger sample size.
To expound the association between PD-L1 expression on immune cells and olaparib efficacy in gBRCA subgroups, we hypothesized a theory of DNA damage accumulation in T cells (Additional file 2: Fig. S3).First, T cells within gBRCAm patients were deficient in BRCA -associated DNA damage repair and exhibited enhanced baseline DNA damage levels compared with BRCA1/2-proficient T cells.Second, PD-L1 expression on immune cells reflected IFNγ-induced adaptive regulation of PD-L1 expression and the presence of pre-existing immune responses including T cell activation [31], which has been demonstrated to accumulate DNA damage [32].Third, PARP inhibition might lead to irreversible T cell death in activated BRCA1/2-deficient T cells, whilst BRCA1/2-proficient T cells will survive PARP inhibition even when they have been activated.Lastly, the anti-tumour efficacy of PARP inhibitors partially depends on T cell activity [9].In summary, PD-L1 expression on immune cells was associated with decreased olaparib efficacy in gBR-CAm patients.In BRCAwt patients, pre-existing T cell activation might indicate an immunoreactive niche that favours olaparib efficacy, and PD-L1 expression on immune cells was correlated with improved olaparib efficacy.Test of interaction revealed that there was statistically significant interaction between PD-L1 expression and gBRCA1/2 mutations or HRR mutation status, whilst no significant interaction was found between PD-L1 expression and HRD status (Additional file 2: Table S14).This hypothesis needs further experimentations to enable reliable validations.
Previous studies have validated the feasibility of ctDNA in detecting mutations of HRR genes in breast and prostate cancers [33][34][35], but relevant studies are scarce in ovarian cancer.Our study obtained high concordance between ctDNA and tumour samples to detect BRCA /HRR mutations.Moreover, ctDNA-derived BRCA / HRR mutations could effectively inform treatment benefits with olaparib.Though ctDNA could accurately detect gBRCA1/2 mutations, its potential to detect somatic BRCA1/2 mutations is unclear and needs to be explored in future studies.The incapability of ctDNA to seek somatic BRCA1/2 mutations might result from the spatial and temporal heterogeneity of somatic BRCA1/2 mutations in a patient's tumour and the rare chances of ovarian cancer to disseminate through the vasculature in contrast to most other types of carcinomas [36][37][38].Due to the limited number of patients whose somatic BRCA1/2 mutations were identified by ctDNA (N = 2), we were unable to correlate the detection of somatic BRCA1/2 mutations with patients' clinical characteristics.
This study had several limitations.First, due to the moderate PD-L1 expression positivity and the proportion of patients with unknown HRD status, the sample size of HRD-negative PD-L1-positive patients was limited (N = 5), which warranted studies with larger patient numbers.Second, the L-MOCA trial was a single-arm study without a placebo-controlled arm.Considering the proven efficacy of olaparib compared with placebo by previous randomised controlled trials in patients with PSROC, the single-arm study design minimises impact on patient survival whilst providing reliable exploratory results.Third, significant features proven to influence olaparib efficacy such as CTLs infiltration were not included in this preplanned exploratory biomarker analysis.Lastly, since only the Asian patients are included in the study, data from other ethnicities are needed to validate the findings.

Table 2
Summary of BRCA and HRR mutation testing results in ctDNA and tumour samples

Table 2
(continued)Patients with more than one mutation origin were summarised in more than one category.Patients with BRCA mutations of undefined origin were counted in 'BRCA1/2' .Patients without mutations or with mutations other than BRCA of undefined origin were counted in 'BRCA wild type' a